During this year we continued some of the projects that were started during the previous year but also initiated new collaborations with several intramural researchers. We continued developing the protocol for high-resolution mapping of the mechanical properties of mouse cartilage matrix by force spectroscopy. The goal is to correlate mechanics with composition maps to further our understanding of function in healthy and osteoarthritic tissue. The issues faced are complex because of the highly inhomogeneous structure and the variable and dense cell population across the tissue depth. The work is now complete and we have shown that we can reliably and robustly map the mechanics through the tissue depth from the joint surface to the bone. We are currently obtaining data for both composition (using infrared spectroscopy) and mechanics on the same tissue slices. The collaboration on the composition and structure of centromeres gave a wealth of data on the dynamic nature of the centromere during the cell cycle. In conjunction with biochemical data we used the AFM to measure heights, shapes and volumes of standard and centromeric nucleosomes that were immunoprecipitated from native chromatin. The centromere was shown to oscillate between the standard octameric structure of nucleosomes to the special form of a tetrameric hemisome right before and during mitosis. We are currently trying to combine AFM with molecular recognition techniques to positively identify the centromere and follow its dynamics. We continued to investigate the role GalR and HU proteins in packaging bacterial DNA and demonstrated that the GalR protein is capable of bridging remote gal operators (GalR binding sites on the DNA) and hence plays a critical role in the 3-D organization of the bacterial chromosome. We next turned our attention to a recent observation that certain noncoding RNAs bind DNA and that the complex interacts with the abundant HU protein. We are currently trying to visualize the DNA conformations resulting by the formation of such ternary complexes. In the past few months we returned to our collaboration investigating the structure and function of retinoschisin (RS) protein, a protein primarily expressed in the retina and shown to be responsible for a form of macular degeneration. Our collaborators have produced what appears to be robust, recombinant RS protein using the baculovirus in insect cells. We succeeded in imaging the protein under ambient conditions and demonstrated the protein to exist as an octameric ring. We are in the process of obtaining a large population of images of such ring structures to that will be used to obtain a high resolution, 3-D reconstruction of the ring. In parallel, we have started looking into the interaction of the protein with artificial, supported lipid bilayers mimicking the composition of the cell membranes of neural cells. A combination of imaging and force spectroscopy will shed light in the role of the protein in maintaining the architecture of the retina. A recent collaboration started from the observation that a mutation in the HMGN5 protein in cell nuclei, also results in heart insufficiency in mice. The hypothesis is that since the protein plays an important role in chromatin packaging, the mutation will result in loser chromatin which may affect the stiffness of the nucleus. This will shift the apparent stiffness of the cells thus impairing the cell capacity during heart cycling. We have developed the protocol to compare the apparent elastic moduli of wildtype and HMGN5 mutant cells to check the hypothesis. We are currently performing measurements to obtain adequate statistics from live cells. Our goal is to be able to measure elasticity changes as a function not only of the presence of the mutant but also as a function of the level of its expression in each cell. Another NCI collaboration aims at visualizing complexes formed by the BRCA1 protein with DNA, supecoiled or linear. Although the role of mutations in breast cancer is known and it capacity to bind DNA is also evident, it appears that the binding is very weak and non-specific. For these reasons establishing the nature of such complexes has been rather elusive. In our effort to visualize the complexes, we use fixation while trying to avoid the obvious artifacts that the method may produce. This is an ongoing project with our collaborators trying to refine their protein production and purification procedures and we test their products and the complexes they produce with DNA.